This invention relates to fraction collection of sample analyte containing solutions. More particularly the present invention is a system and method which allows user controlled formation and ejection of small volume droplets or streams of sample analyte containing solutions, thereby enabling precise user directed fractionalization and distributed collection of sample analyte(s).
Chromatographic separation systems for separating fractions of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), oligonucleotides, RNA and the like by chromatography have relied upon collection carousels to collect the samples. In one conventional system, a carousel positions collection vials, one after the other, under the outlet of the separation equipment for a specified length of time.
A new chromatographic separation process, Matched Ion Pair Chromatography (MIPC), has presented a separation process which can separate dsDNA into base-length sized fractions with an unprecedented precision. Each base-pair length is eluted in a predictable and calculable solvent concentration, permitting a user to precisely predict the specific time when a fraction having a particular base-pair length will be delivered from the bottom opening of the separation column. A new chromatographic separation system described in Provisional Application Serial No. 60/119,936 filed Feb. 12, 1999 and applications cited therein, has been constructed to apply the MIPC separation technology. To make full use of this new technology, eluant from the separation column containing a specific target fraction or fractions must be collected, unmixed, in separate identified vials. The prior art collection systems are unable to satisfy this need.
A most common chromatography eluant collection system uses a circular carousel with a circular array of receptors which contain sample collection vials. Modern biochemical procedures involve simultaneous processing a large number of samples, and rectangular multiwell or microtiter plates or trays with 96, 384 and 1536 sample well or vial configurations with standardized dimensions and X-axis and Y-axis positioning of each well are in common use. It is desirable to collect fractions from the MIPC separation systems directly into designated respective vials or wells in these collection plates. The traditional systems are unable to satisfy this need.
Two-way valves are used in many systems to redirect liquid flow to a by-pass or shunt. However, valves of this type are unsatisfactory for diverting the fractions into collecting vials because they introduce xe2x80x9cdead-volumexe2x80x9d which degrades the separation between fractions and causes contamination from one fraction to the next.
Prior art systems also produce droplets having a volume which may be so large as to include more than the segment to be collected. Reducing droplet size by using a smaller outlet orifice can introduce shear forces which can disrupt or break larger polynucleotides.
One object of this invention is an apparatus and method for collecting with precision an eluant fraction from a chromatographic column into a designated collection vial at a predetermined time.
It is a further object of this invention to provide an apparatus and method for collecting with precision eluant fractions from a chromatographic column into designated vials of a multiwell plate.
It is a still further object of this invention to provide a system for enabling a drop-by-drop ejection of sample analyte containing solution from a contained flow stream thereof into a collection vial with small volumes and without subjecting the liquid to destructive shear forces.
It is another object of this invention to provide a system for separating an aqueous stream of mixed polynucleotides into a series of length-based polynucleotide fractions and collecting one or more of the length-based polynucleotide fractions into separate containers. The system comprises a separation column containing separation media for separating an aqueous stream of mixed polynucleotides into a series of length-based polynucleotide fractions; a container including one or more single-sample vials or wells; an ejection chamber having a separated sample inlet for receiving the length-based polynucleotide fractions, a waste outlet for discharging uncollected sample, and a capillary-sized fraction outlet positioned to discharge a selected length-based polynucleotide fraction into a single-sample container. The system also includes means for effecting discharge of a selected length-based polynucleotide fraction into the separate container.
The means for effecting discharge of a selected length-based polynucleotide fraction into a separate container can include a puff valve having a pressurized gas inlet and a puff gas outlet and the ejection chamber can include a puff gas inlet communicating with the puff gas outlet, whereby activation of the puff valve will discharge a puff of gas into the ejection chamber and will effect discharge of liquid sample through the fraction outlet.
The means for effecting discharge of a selected length-based polynucleotide liquid fraction into a separate container can include a flow restriction actuator, a flow restriction in the waste conduit which will restrict flow of uncollected sample upon actuation by the flow restriction actuator, whereby actuation of the flow restriction will effect an increase in liquid pressure in the ejection chamber. Actuation of the flow restriction can effect discharge of sample through the fraction outlet. The system preferably includes computer control means for effecting discharge of the length-based polynucleotide fractions by actuation of the flow restriction. This can be combined with a puff valve having a pressurized gas inlet and a puff gas outlet wherein the ejection chamber includes a puff gas inlet communicating with the puff gas outlet, whereby activation of the puff valve will discharge a puff of gas into the ejection chamber and will effect discharge of sample through the fraction outlet. In this combination, the computer control means can effect discharge of the length-based polynucleotide fractions by opening the air-puff valve.
The fraction outlet has an outlet opening, and the outlet opening can be combined with drop size reduction means for reducing the size of droplets discharged into the sample container. The drop size reduction means can be a gas-knife surrounding the fraction outlet and positioned to dislodge fluid through the outlet opening in the form of small droplets; a piezo-electric vibrator, or an electrostatic separator, or a combination thereof. The drop size reduction means can include a nozzle having a small orifice wherein the gas-knife surrounds the nozzle and is positioned to dislodge fluid through the outlet opening in the form of small droplets; the drop size reduction means can be a piezo-electric vibrator positioned adjacent to the nozzle; or the drop size reduction means can be an electrostatic separator, and the nozzle having a charge opposite to the charge of the sample container.
The system of this invention can include computer control means for controlling the means for effecting discharge of the length-based polynucleotide fractions. The computer control means can include means for responding to a fraction detector output signal to determine the time interval for effecting discharge of a length-base fraction, means for responding to a fraction detector output signal to determine when the signal strength exceeds a threshold value to determine the time interval for effecting discharge of a length-base fraction, or means for responding to a fraction detector output signal to determine when the signal slope exceeds a preset value to determine the time for beginning discharge of a length-base fraction.
The method of this invention includes separating an aqueous stream of mixed polynucleotides into a series of length-based polynucleotide fractions and collecting one or more of the length-based polynucleotide fractions into separate single-fraction containers with a system comprising a chromatographic separation system for separating a mixture of polynucleotides into size-based fractions in an eluant stream; an ejection chamber having a separated sample inlet for receiving the length-based polynucleotide fractions, a waste outlet for discharging uncollected sample, and a fraction outlet positioned to discharge a selected length-based polynucleotide fraction into a single-sample container; and means for effecting discharge of a selected length-based polynucleotide fraction into the single-sample container. The method comprises the steps of (a) passing the eluant stream carrying successive, separated size-based fractions through the ejection chamber; (b) determining when a selected length-based polynucleotide fraction will pass through the ejection chamber; and (c) effecting discharge of a selected length-based polynucleotide fraction into a sample container.
In this method, the time interval within which a selected length-based polynucleotide fraction will pass through the ejection chamber is determined, and eluant in the ejection chamber during this time interval is discharged into the sample container. When the system includes a detector system between the separation system and the ejection chamber and the detector system produces an output signal which is a function of the concentration of a fraction in the eluant stream, step (b) can comprise determining when the output signal from the detector exceeds a preset threshold level indicating the presence of the selected fraction; or determining when the output signal from the detector exceeds a preset slope indicating the presence of the selected fraction.